We study the regulation of amino acid and vitamin biosynthetic genes in budding yeast as a means of dissecting mechanisms of translational and transcriptional control of gene expression. Transcription of these genes is coordinately induced by transcriptional activator Gcn4 in response to amino acid limitation. Gcn4 expression is coupled to amino acid levels through a translational control mechanism that mediates increased synthesis of Gcn4 under starvation conditions in which general protein synthesis is down-regulated. We showed previously that efficient transcriptional activation by Gcn4 depends on recruitment of coactivator complexes Mediator, SAGA, SWI/SNF, and RSC, which collectively mediate nucleosome remodeling and recruitment of general transcription factors and RNA Polymerase II (Pol II) to promoters. We further demonstrated that SAGA is recruited co-transcriptionally to coding sequences by association with the Ser5-phosphorylated C-terminal domain (CTD) of Pol II (Ser5P), and that the histone acetyltransferase (HAT) subunit of SAGA (Gcn5) promotes increased histone acetylation, histone eviction, Pol II processivity, and histone H3-Lys4 methylation within coding sequences. Recently, we demonstrated that the histone H4 HAT complex, NuA4, is also recruited co-transcriptionally to coding regions in a manner stimulated by the Ser5 CTD kinase Cdk7/Kin28, and that NuA4 association with nucleosomes further depends on H3 methylation, presumably due to chromodomains and a PHD finger in NuA4 subunits. We also obtained evidence that the HAT activities of NuA4 and SAGA cooperate to enhance co-transcriptional recruitment of the nucleosome remodeler RSC, promote histone eviction from transcribed sequences, and stimulate Pol II elongation rate. Consequently, inactivating Gcn5 and the HAT subunit in NuA4 (Esa1) confers additive reductions in transcript production from long versus short coding sequences in vivo. These findings demonstrate direct, additive roles for the HAT activities of NuA4 and SAGA in promoting the elongation phase of II transcription. We recently extended the two-stage recruitment mechanism, via Ser5P and methylated histones, described above for NuA4 to include the histone deacetylase complex (HDAC) Rpd3C(S). It was known that methylation of H3 by Set1 and Set2 is required for deacetylation of coding region nucleosomes by Set3C and Rpd3C(S), respectively. We discovered that Set3C and Rpd3C(S) are co-transcriptionally recruited to coding sequences in the absence of both Set1 and Set2, but in a manner stimulated by Cdk7/Kin28. Moreover, Rpd3C(S) and Set3C were shown to interact with both Pol II-Ser5P and histones in extracts, but only the histone interactions require H3 methylation. Moreover, a reconstituted Rpd3C(S) complex bound specifically to Ser5P synthetic peptides. Thus, whereas interaction with methylated H3 is required for Rpd3C(S) and Set3C deacetylation activities, their co-transcriptional recruitment is stimulated by the phosphorylated Pol II CTD. We further demonstrated that the HDAs Rpd3, Hos2, and Hda1 have overlapping functions in deacetylating nucleosomes and limiting co-transcriptional nucleosome eviction. Moreover, a strong correlation between increased acetylation and lower histone occupancy observed in single, double, and triple HDA mutants supports our contention, based on analysis of HAT mutants, that histone acetylation is a key determinant of co-transcriptional nucleosome eviction. The cyclin-dependent kinase (Cdk) Bur1/Bur2 is the yeast ortholog of mammalian P-TEFb, which phosphorylates Ser2 of the Pol II CTD, but its role in CTD phosphorylation in yeast cells was unclear. We recently demonstrated that Bur1/Bur2 is co-transcriptionally recruited to the 5 end of Gcn4 target gene ARG1 in a manner stimulated by interaction of the Bur1 C-terminus with the Ser5P-CTD. Furthermore, impairing Bur1/Bur2 or removing the CTD-interaction domain in Bur1 reduces Ser2P in bulk elongating Pol II phosphorylated on Ser5 and in Pol II near the 5 end of ARG1 to nearly the same extent as does eliminating the major Ser2 CTD kinase Ctk1. By contrast, Ctk1 is responsible for the bulk of Ser2P-CTD in total Pol II and at promoter-distal sites. These and other findings indicate that CTD phosphorylation involves a cascade of Cdk function, wherein Bur1/Bur2 is recruited to promoters by Ser5P-CTD generated by Kin28, phosphorylates Ser2 near promoters, and stimulates Ser2-CTD phosphorylation at promoter-distal sites by Ctk1 The Mediator is a multisubunit coactivator required for transcription initiation by Pol II, which is recruited by Gcn4 to its target promoters in vivo. Previously, we determined that the tail subdomain of Mediator, containing the Gal11/Med15 subunit, is a direct target of Gcn4 in vivo, critical for recruitment by Gcn4 of both intact Mediator and the stable Mediator tail subdomain existing in sin4&#61508;cells. Although several Gal11 segments were shown previously to bind Gcn4 in vitro, the importance of these interactions for recruitment of Mediator and transcriptional activation by Gcn4 in cells was unknown. We demonstrated that interaction of Gcn4 with the Mediator tail in vitro, and recruitment of this subcomplex and intact Mediator to the ARG1 promoter in vivo, involve additive contributions from three different segments in the N-terminus of Gal11. These include the KIX domain, which is a critical target of other activators, and a region that shares a conserved motif (B-box) with mammalian coactivator SRC-1, and we established that the B-box is a critical determinant of Mediator recruitment by Gcn4. We further demonstrated that Gcn4 binds to the Gal11 KIX domain directly and, in collaboration with Christopher Jaroniecs group at Ohio State University, utilized NMR chemical shift analysis, combined with mutational studies, to identify the likely Gcn4 binding site on the surface of the KIX domain. Together, these results define the physiological mechanism of Mediator recruitment by Gcn4. It appears that Gcn4 is distinctive in relying on comparable contributions from multiple segments of Gal11 for efficient recruitment of Mediator to target promoters in vivo. The hnRNP protein Npl3 of budding yeast has dual functions in promoting transcription elongation and preventing termination at cryptic termination sites by Pol II. Npl3 was known to be a substrate of arginine methyltransferase Hmt1, but the role of Hmt1 in regulating Npl3s functions in transcription antitermination and elongation were unknown. In collaboration with Chi-Ming Wong and colleagues at the University of Hong Kong, we found that mutants lacking Hmt1 methyltransferase activity exhibit reduced recruitment of Npl3, but elevated recruitment of a component of mRNA cleavage/termination factor CFI, to the transcriptionally activated GAL10-GAL7 locus. Consistent with this, hmt1 mutants displayed increased termination at the defective gal10-&#61508;56 terminator, indicating a reduction in antitermination activity. Remarkably, hmt1 cells also exhibit diminished recruitment of transcription elongation factor Tho2 (a component of the THO complex) and a reduced rate of transcription elongation in vivo. Importantly, the defects in recruitment of Npl3 and Tho2, in antitermination, and in transcription elongation in hmt1&#61508;cells were all mitigated by substitutions in the RGG repeats of Npl3 that functionally mimic arginine methylation by Hmt1. These findings indicate that Hmt1 promotes transcription elongation and suppresses utilization of cryptic termination sites by methylating the RGG repeats in Npl3. As Hmt1 stimulates dissociation of Tho2 from an Npl3-mRNP complex, it might act to recycle these factors back to sites of ongoing transcription as the means of promoting their functions in transcription elongation and antitermination.